Penicillin streptomycin 100

Murine smooth muscle cells (SMCs) were isolated from the intimal–medial layer of aortae of C57BL/6 mice of both sexes (The Jackson Lab, Bar Harbor, ME, USA) as described in [11 (link)]. Subconfluent SMCs were incubated in DMEM (Euroclone, Milan, Italy) supplemented with 0.2% essential-fatty-acid-free albumin.
Human aortic SMCs (PCS-100-012, ATCC, MA, USA) were cultured in ATCC Vascular Cell Basal Medium (PCS-100-030, ATCC; 500 mL supplemented with 500 µL ascorbic acid, 500 µL rh EGF, 500 µL rh insulin and rh FGF-b, 25 mL glutamine), 5% FBS (ATCC Vascular Smooth Muscle Growth kit), and 5 mL penicillin–streptomycin 100× (Euroclone, Milan, Italy).

As we published before [14 (link)], in order to induce antibody production from MBCs, we polyclonally stimulated PBMCs with CpG. Briefly, PBMCs were cultured in complete medium at a concentration of 1 × 10⁶ cells/mL. Complete medium was prepared as follows: RPMI-1640 (Euroclone, Milan, Italy), 10% heat inactivated HyClone FBS (Hyclone Laboratories, Logan, UT, USA), 1mM L-Glutamine (GIBCO BRL, New York, NY, USA); 1× Penicillin/Streptomycin 100× (Euroclone, Milan, Italy), 1% sodium pyruvate (GIBCO BRL, New York, NY, USA). Cells were stimulated for 5 days with 0.35 µM TLR9 agonist CpG-B ODN2006 (Hycult Biotech, Uden, The Netherland) plus 20 ng/mL rhIL-21 (Milteny, Bergisch Gladbach, Germany) and 20 ng/mL rhIL-4 (Milteny, Bergisch Gladbach, Germany).

The six cell lines, A375, H460, HT29, MCF7, HepG2, and HaCaT (Lonza, Verviers, Belgium), were kept in culture and expanded at 37 °C in a humidified atmosphere of 5% CO₂ in DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza, Basel, Switzerland) culture medium for HaCaT, HepG2, HT29, MCF7, and A375, and RPMI-1640 for H460, supplemented with 10% FBS, Penicillin/Streptomycin 100× (Euroclone, Devon, UK), Glutamax 100× (Invitrogen, Carlsbad, CA, USA) and non-essential amino acids 100× (Invitrogen). Phosphate buffer (PBS, phosphate-buffered saline, Ca²⁺ and Mg²⁺ free) and trypsin (Ca²⁺ and Mg²⁺ free) were supplied by Euroclone. The cells were plated 15 × 10³ perwell in 96-well tissue culture plates and allowed to attach for 24 h. Then, cells were treated with PECS dissolved in culture medium supplemented with 1% FBS at different concentrations for 48 h (50 μg/mL, 80 μg/mL, 110 μg/mL, 140 μg/mL, 170 μg/mL, and 200 μg/mL). PECS were dissolved in sterile H₂O at 100 mg/mL.